Cancer-associated Fibroblasts Activation Research Articles

Abstract PR-006: Pulsed low-dose-rate radiation (PLDR) limits pancreatic pro-tumor stroma aggravation: Pre-clinical basis for an ongoing PLDR trail

Abstract Radiation, combined with radio-sensitizing chemotherapy, is used preoperatively as neoadjuvant therapy (NT) for borderline resectable pancreatic ductal adenocarcinoma (PDAC) with the intent of facilitating a curative surgical intervention. PDAC is uniquely characterized by an extensive fibrous microenvironment, desmoplasia, which can unintendedly be aggravated by NT to foster its pro-tumoral function. Radiation, as part of NT, is aimed at providing a margin adjacent to un-resectable vessels and sterilizing regional lymph nodes. Yet, to avoid the unintended desmoplastic aggravation, and because of a risk of toxicities associated with high doses due to the radio-sensitivity of adjacent small bowel and stomach, the total dose of NT radiation delivered to PDAC patients is classically modest. Therefore, NT radiation in PDAC patients is often lower than optimal for effective tumor cell elimination. Pulsed low-dose-rate (PLDR) radiation improves the safety of radiation treatment as it allows time for DNA damage repair in non-tumorous cells/tissues while simultaneously remaining as effective as continuous dose rate (CDR) radiation in cancer cells. In theory, PLDR could be amendable for increased radiation dosage. Of interest, the use of PLDR in pre-clinical animal studies revealed a systemic lowering of transforming growth factor beta (TGFβ), a known immunosuppressive factor. Hence, we posit that since cancer-associated fibroblasts (CAFs) produce TGFβ and constitute one of the most abundant cells in PDAC desmoplasia, variations in TGFβ levels in response to NT will inform on desmoplastic dynamic changes. Our team has developed means to assess CAF functions and activation statuses. These efforts were guided by generating a desmoplastic biomarker signature obtained from patient-harvested CAFs during the production of extracellular matrix (ECM), using our well-established in vitro 3D system. We employed this in vivo-mimetic system to test the hypothesis that PLDR limits pro-tumoral desmoplastic CAF aggravation. As part of the CAF functional signature, we tested levels of immunosuppressive TGFβ secretion, expression of netrin-G1, palladin, and others; together with the biogenesis of unique extracellular vesicles, and the production of ECMs capable of nurturing PDAC cells under starvation. During a 5-day assay, human PDAC 3D ECM producing CAFs were treated with gemcitabine (Gem; 5nm) plus 4Gy or 8Gy PLDR vs. CDR. Results indicated that Gem alone or with CDR indeed aggravated CAF’s pro-PDAC phenotype and function, while Gem combined with PDLR limited and sometimes reverted this functional pro-tumor CAF signature. Based on these results, we are poised to test this functional CAF/desmoplasia signature, as laboratory correlatives, in an ongoing clinical trial at Fox Chase Cancer Center. The phase I trial is a dose-escalation study of PLDR radiation and chemotherapy at standard and intensified doses in preoperative pancreatic cancer patients with toxicity, histopathologic, and CAF-informing translational endpoints. Citation Format: Janusz Franco-Barraza, Tiffany Luong, Jessica K. Wong, Debora B. Vendramini-Costa, Ralph Francescone, Jaye C. Gardiner, Kristopher S. Raghavan, Joshua E. Meyer, Edna Cukierman. Pulsed low-dose-rate radiation (PLDR) limits pancreatic pro-tumor stroma aggravation: Pre-clinical basis for an ongoing PLDR trail [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-006.

Abstract PR-001: Angiotensin II receptor blockers modify the solid tumor microenvironment and improve radiation therapy response

Abstract Poorly oxygenated (hypoxic) cells are key components of the solid tumor microenvironment and are well-established contributors to poor therapeutic outcome. Patients with tumors that contain hypoxic cells have worse outcome after radiotherapy, chemotherapy, and surgery, underscoring the therapeutic relevance of hypoxia in driving more aggressive tumor phenotypes. Angiotensin II type 1 receptor (AT1R) blockers (ARBs) are widely prescribed anti-hypertensive agents that inhibit the activity of angiotensin II derived from the renin-angiotensin system. ARBs have also been shown to exhibit anti-fibrotic activity by inhibiting AT1R expressed on myofibroblasts. We recently reported that the ARB telmisartan inhibits collagen I (Col1) deposition in solid tumors, increasing net tumor perfusion, stabilizing microregional tumor blood flow, and decreasing tumor hypoxia. Cancer-associated fibroblasts (CAFs) produce Col1, although recent clinical data indicate the presence of multiple, functionally-diverse CAF subsets in tumors from patients with breast or ovarian cancer. To further explore how ARBs and CAFs affect the solid tumor microenvironment, we were interested in whether murine CAF subsets are present in human tumor xenografts and, if so, whether telmisartan preferentially influences collagen-producing CAF subsets in solid tumors. We were also interested in whether telmisartan affects collagen deposition, hypoxia, and radiation response in head & neck and cervix tumors that are commonly treated with ionizing radiation therapy. We then asked whether head & neck cancer patients taking ARBs as anti-hypertensive medications have quantifiably different tumor microenvironments and improved therapeutic outcome. Using a panel of six human head & neck and cervix tumor xenografts, we identified four unique subsets (S1-S4) of CAFs that are present at varying levels in the different tumor lines. Telmisartan significantly altered the CAF content of tumors while also reducing CAF-mediated collagen deposition in the tumor microenvironment. We found that telmisartan also decreased tumor hypoxia and improved radiation response in our pre-clinical models, indicating that CAF subsets help to create hypoxia and contribute to radioresistance of solid tumors in an AT1R-dependent manner. Using a retrospective cohort of over 1,100 oropharyngeal cancer patients, we found that patients taking ARBs had dramatically improved therapeutic outcomes after radiation therapy, validating our pre-clinical data and supporting the further development and testing of ARBs as neoadjuvant therapies for patients with hypoxic tumors. Understanding how the solid tumor microenvironment influences cancer therapy is central to improving treatment outcome, and our work identifies CAFs as key contributors to the development of hypoxia in solid tumors. Repurposing clinically approved, anti-hypertensive ARBs represents a novel therapeutic strategy to inhibit CAF activity, modify the solid tumor microenvironment, reduce tumor hypoxia, and improve radiation response. Citation Format: Brennan J. Wadsworth, Che-Min Lee, Ryan Urban, Sarah N. Hamilton, Kevin L. Bennewith. Angiotensin II receptor blockers modify the solid tumor microenvironment and improve radiation therapy response [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-001.

Cancer-associated fibroblasts activated by miR-196a promote the migration and invasion of lung cancer cells

Fibroblasts in the tumor microenvironment, known as cancer-associated fibroblasts (CAFs), promote the migration, invasion, and metastasis of cancer cells when they are activated through diverse processes, including post-transcriptional regulation by microRNAs (miRNAs). To identify the miRNAs that regulate CAF activation, we used NanoString to profile miRNA expression within normal mouse lung fibroblasts (LFs) and CAFs. Based on NanoString profiling, miR-196a was selected as a candidate that was up-regulated in CAFs. miR-196a-overexpressed LFs (LF-196a) promoted the migration and invasion of lung cancer cells in co-culture systems (Transwell migration and spheroid invasion assays). ANXA1 was confirmed as a direct target of miR-196a, and adding back ANXA1 to LF-196a restored the cancer cell invasion promoted by miR-196a. miR-196a increased CCL2 secretion in fibroblasts, and that was suppressed by ANXA1. Furthermore, blocking CCL2 impeded cancer spheroid invasion. In lung adenocarcinoma patients, high miR-196a expression was associated with poor prognosis. Collectively, our results suggest that CAF-specific miR-196a promotes lung cancer progression in the tumor microenvironment via ANXA1 and CCL2 and that miR-196a will be a good therapeutic target or biomarker in lung adenocarcinoma.

Interfacial Curvature in Confined Coculture Directs Stromal Cell Activity with Spatial Corralling of Pancreatic Cancer Cells.

Interfacial cues in the tumor microenvironment direct the activity and assembly of multiple cell types. Pancreatic cancer, along with breast and prostate cancers, is enriched with cancer-associated fibroblasts (CAFs) that activate to coordinate the deposition of the extracellular matrix, which can comprise over 90% of the tumor mass. While it is clear that matrix underlies the severity of the disease, the relationship between stromal-tumor cell assembly and cell-matrix dynamics remains elusive. Micropatterned hydrogels deconstruct the interplay between matrix stiffness and geometric confinement, guiding heterotypic cell populations and matrix assembly in pancreatic cancer. Interfacial cues at the perimeter of microislands guide CAF migration and direct cancer cell assembly. Computational modeling shows curvature-stress dependent cellular localization for cancer and CAFs in coculture. Regions of convex curvature enhance edge stress that activates a myofibroblast phenotype in the CAFs with migration and increased collagen I deposition, ultimately leading to a central "corralling" of cancer cells. Inhibiting mechanotransduction pathways decreases CAF activation and the associated corralling phenotype. Together, this work reveals how interfacial biophysical cues underpin aspects of stromal desmoplasia, a hallmark of disease severity and chemoresistance in the pancreatic, breast, and prostate cancers, thereby providing a tool to expand stroma-targeting therapeutic strategies.

TGF-β1-activated cancer-associated fibroblasts promote breast cancer invasion, metastasis and epithelial-mesenchymal transition by autophagy or overexpression of FAP-α

Cancer-associated fibroblasts (CAFs) play an important role in the initiation, metastasis, and invasion of breast cancer. However, whether autophagy acts as a tumor promotion mechanism by inducing epithelial-mesenchymal transition (EMT) is still controversial and remains undefined at the mechanistic levels. In this study, we investigated whether autophagy or FAP-α is required for the invasion, pulmonary metastasis and EMT of breast cancer cells and underlying mechanism. We employed an in vitro model of NIH3T3 fibroblasts treated with H2O2 and confirmed that TGF-β1 could convert fibroblasts into CAFs through autophagy under oxidative stress in the tumor microenvironment. Modulation of autophagy by rapamycin, 3-methyladenine or ATG-5 knockdown regulated the expression of CAFs markers, suggesting a role of autophagy in the tumor promotion mechanism of TGF-β1-induced CAFs activation. Furthermore, we established an indirect co-culture model and a mixed xenograft as a corresponding in vivo model. We demonstrated that TGF-β1-activated CAFs promote tumor invasion, pulmonary metastasis and EMT, which act through autophagy and overexpression of FAP-α in both models, while autophagy inhibitor 3-methyladenine blocked these effects induced by TGF-β1-activated CAFs. Moreover, the co-localization of LC3β and EMT marker vimentin in mixed xenograft also revealed that TGF-β1-activated CAFs promote tumor growth, pulmonary metastasis, and EMT program partly through autophagy. In addition, knockdown of FAP-α resulted in reversed EMT and abolished tumor invasion and pulmonary metastasis induced by TGF-β1-activated CAFs. Taken together, we conclude that both autophagy and FAP-α are required for breast cancer cell invasion and metastasis. Targeting autophagy or FAP-α rather than both can serve as a potential approach to improve the prognosis for human breast cancer.

RETRACTED ARTICLE: MiR-151-3p transferred by cancer-associated fibroblast-derived extracellular vesicles promotes osteosarcoma progression through the CHL1/integrin 1\u03b2/TGF-\u03b2 axis

microRNA-151-3p (miR-151-3p) is a widely reported oncogene with a documented role in tumorigenesis and cancer progression. miRNAs are potent regulators of gene expression that are transferred between cells by extracellular vesicles (EVs). However, there is limited data about the role of EV-derived miR-151-3p in osteosarcoma (OS) progression, or the possible effects of miR-151-3p on the paracrine activity of cancer-associated fibroblasts (CAFs) in OS. To fill this gap of knowledge, we isolated CAF-derived EVs (CAF-EVs) from examined their up-take by human MG63 OS cells and their effects on MG63 biology. We found high expression of miR-151-3p in OS tissues, and miR-151-3p derived from CAF-EVs promoted the process of epithelial-mesenchymal transition (EMT), migration, and invasion of MG63 cells. Notably, bioinformatics analysis, RIP, and dual luciferase report test determined that CHL1 was the direct binding target of miR-151-3p. CAF-EVs mediated the CHL1/integrin 1β axis through miR-151-3p to regulate the TGF-β pathway and then promoted the proliferation, migration, invasion, and EMT of OS cells. Our in vivo results confirmed that EV secretion of miR-151-3p activated the TGF-β pathway through the CHL1/integrin 1β axis to promote the proliferation of OS cells and increase tumor volume and weight. Other effects were to upregulate E-Cadherin and downregulate the expression of N-Cadherin and β-catenin. Hence, our data reveal a potential functional axis mediated by EVs in the tumor microenvironment of OS and suggest potential candidate therapy targets.

Palladin isoforms 3 and 4 regulate cancer-associated fibroblast pro-tumor functions in pancreatic ductal adenocarcinoma

Pancreatic Ductal Adenocarcinoma (PDAC) has a five-year survival under 10%. Treatment is compromised due to a fibrotic-like stromal remodeling process, known as desmoplasia, which limits therapeutic perfusion, supports tumor progression, and establishes an immunosuppressive microenvironment. These processes are driven by cancer-associated fibroblasts (CAFs), functionally activated through transforming growth factor beta1 (TGFβ1). CAFs produce a topographically aligned extracellular matrix (ECM) that correlates with reduced overall survival. Paradoxically, ablation of CAF populations results in a more aggressive disease, suggesting CAFs can also restrain PDAC progression. Thus, unraveling the mechanism(s) underlying CAF functions could lead to therapies that reinstate the tumor-suppressive features of the pancreatic stroma. CAF activation involves the f-actin organizing protein palladin. CAFs express two palladin isoforms (iso3 and iso4) which are up-regulated in response to TGFβ1. However, the roles of iso3 and iso4 in CAF functions remain elusive. Using a CAF-derived ECM model, we uncovered that iso3/iso4 are required to sustain TGFβ1-dependent CAF activation, secrete immunosuppressive cytokines, and produce a pro-tumoral ECM. Findings demonstrate a novel role for CAF palladin and suggest that iso3/iso4 regulate both redundant and specific tumor-supportive desmoplastic functions. This study highlights the therapeutic potential of targeting CAFs to restore fibroblastic anti-tumor activity in the pancreatic microenvironment.

Cancer-Associated Fibroblasts Suppress Cancer Development: The Other Side of the Coin.

Cancer-associated fibroblasts (CAFs) are the main stromal components of cancer, representing a group of heterogeneous cells. Many studies indicate that CAFs promote tumor development. Besides, evidence of the tumor suppression effects of CAFs keeps on merging. In the tumor microenvironment, multiple stimuli can activate fibroblasts. Notably, this does not necessarily mean the activated CAFs become strong tumor promoters immediately. The varying degree of CAFs activation makes quiescent CAFs, tumor-restraining CAFs, and tumor-promoting CAFs. Quiescent CAFs and tumor-restraining CAFs are more present in early-stage cancer, while comparatively, more tumor-promoting CAFs present in advanced-stage cancer. The underlying mechanism that balances tumor promotion or tumor inhibition effects of CAFs is mostly unknown. This review focus on the inhibitory effects of CAFs on cancer development. We describe the heterogeneous origin, markers, and metabolism in the CAFs population. Transgenetic mouse models that deplete CAFs or deplete CAFs activation signaling in the tumor stroma present direct evidence of CAFs protective effects against cancer. Moreover, we outline CAFs subpopulation and CAFs derived soluble factors that act as a tumor suppressor. Single-cell RNA-sequencing on CAFs population provides us new insight to classify CAFs subsets. Understanding the full picture of CAFs will help translate CAFs biology from bench to bedside and develop new strategies to improve precision cancer therapy.

Stromal-driven and Amyloid \u03b2-dependent induction of neutrophil extracellular traps modulates tumor growth

Tumors consist of cancer cells and a network of non-cancerous stroma. Cancer-associated fibroblasts (CAF) are known to support tumorigenesis, and are emerging as immune modulators. Neutrophils release histone-bound nuclear DNA and cytotoxic granules as extracellular traps (NET). Here we show that CAFs induce NET formation within the tumor and systemically in the blood and bone marrow. These tumor-induced NETs (t-NETs) are driven by a ROS-mediated pathway dependent on CAF-derived Amyloid β, a peptide implicated in both neurodegenerative and inflammatory disorders. Inhibition of NETosis in murine tumors skews neutrophils to an anti-tumor phenotype, preventing tumor growth; reciprocally, t-NETs enhance CAF activation. Mirroring observations in mice, CAFs are detected juxtaposed to NETs in human melanoma and pancreatic adenocarcinoma, and show elevated amyloid and β-Secretase expression which correlates with poor prognosis. In summary, we report that CAFs drive NETosis to support cancer progression, identifying Amyloid β as the protagonist and potential therapeutic target.

Autophagy is deregulated in cancer-associated fibroblasts from oral cancer and is stimulated during the induction of fibroblast senescence by TGF-β1

Many of the characteristics ascribed to cancer-associated fibroblasts (CAFs) are shared by activated, autophagic and senescent fibroblasts. Whilst most oral squamous cell carcinomas (OSCCs) are genetically unstable (GU-OSCC), genetically stable variants (GS-OSCC) have been described and, notably, CAF activation (myofibroblast differentiation) and senescence are characteristics particularly associated with GU-OSCCs. However, it is not known whether autophagy is disrupted in these cells or whether autophagy regulates the development of the myofibroblast and senescent phenotypes. In this study, we show that senescent CAFs from GU-OSCCs contained more autophagosomes than normal human oral fibroblasts (NHOFs) and CAFs from GS-OSCCs possibly due to autophagic impairment. Further, we show that deregulation of autophagy in normal fibroblasts, either by inhibition with autophagy inhibitor, SAR405, or activation with TGF-β1, induced fibroblast activation and senescence: In response to TGF-β1, autophagy was induced prior to the development of the activated and senescent phenotypes. Lastly, we show that both SAR405- and TGF-β1-treated NHOFs enhance OSCC cell migration but only TGF-β1-treated cells increase OSCC invasion through Matrigel, indicating that TGF-β1 has additional effects that are independent of fibroblast activation/senescence. These results suggest a functional role for autophagy in the development of myofibroblast and CAF phenotypes.

DNA methylation mediated down-regulation of ANGPTL4 promotes colorectal cancer metastasis by activating the ERK pathway.

Background: Colorectal cancer (CRC) imposes significant health burden and is increasing in incidence. NGPTL4 has been implicated in the development of CRC. The present study aimed to investigate the molecular mechanisms by which ANGPTL4 expression might regulate epithelial-mesenchymal transition (EMT) and the tumor microenvironment in CRC.Methods: CRC and para-carcinoma tissues were collected from 67 CRC patients. ANGPTL4 expression levels and DNA methylation of ANGPTL4 promoter region were determined. Next, the migration and invasion capacities of CRC cells were assessed. Immunofluorescence and Western blot were used to identify the signaling pathways by which ANGPTL4 mediated tumor metastasis. A tumorigenesis mice model with transplanted fibroblast cells and ANGPTL4 overexpressed CRC cells was established to investigate the effects of ANGPTL4 on the metastasis of cancer cells in vivo.Results: ANGPTL4 was significantly decreased in CRC tissues and DNA hypermethylation was involved in the regulation of ANGPTL4. Mechanistically, ANGPTL4 induced activation of cancer-associated fibroblasts in the tumor microenvironment and promoted EMT in CRC cells through the ERK signaling pathway. In vivo, the overexpression of ANGPTL4 was found to inhibit the metastasis of tumor cells in lung tissues.Conclusion: DNA hypermethylation induced ANGPTL4 downregulation promoted the activation of cancer-associated fibroblasts and epithelial mesenchymal transformation of CRC cells via the ERK signaling pathway, thereby promoting invasion and metastasis in CRC.

Snail1 expression in endothelial cells controls growth, angiogenesis and differentiation of breast tumors.

Snail1 is a transcriptional factor required for epithelial to mesenchymal transition and activation of cancer-associated fibroblasts (CAF). Apart from that, tumor endothelial cells also express Snail1. Here, we have unraveled the role of Snail1 in this tissue in a tumorigenic context.Methods: We generated transgenic mice with an endothelial-specific and inducible Snail1 depletion. This murine line was crossed with MMTV-PyMT mice that develop mammary gland tumors and the consequence of Snail1 depletion in the endothelium were investigated. We also interfere Snail1 expression in cultured endothelial cells.Results: Specific Snail1 depletion in the endothelium of adult mice does not promote an overt phenotype; however, it delays the formation of mammary gland tumors in MMTV-PyMT mice. These effects are associated to the inability of Snail1-deficient endothelial cells to undergo angiogenesis and to enhance CAF activation in a paracrine manner. Moreover, tumors generated in mice with endothelium-specific Snail1 depletion are less advanced and show a papillary phenotype. Similar changes on onset and tumor morphology are observed by pretreatment of MMTV-PyMT mice with the angiogenic inhibitor Bevacizumab. Human breast papillary carcinomas exhibit a lower angiogenesis and present lower staining of Snail1, both in endothelial and stromal cells, compared with other breast neoplasms. Furthermore, human breast tumors datasets show a strong correlation between Snail1 expression and high angiogenesis.Conclusion: These findings show a novel role for Snail1 in endothelial cell activation and demonstrate that these cells impact not only on angiogenesis, but also on tumor onset and phenotype.

Acidic tumor microenvironment-sensitive liposomes enhance colorectal cancer therapy by acting on both tumor cells and cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) play a crucial role in facilitating tumor invasion and metastasis, which act as the "soil" in the tumor microenvironment (TME). Accordingly, it would be a promising strategy to enhance the antitumor effect by killing both tumor cells and CAFs simultaneously. Herein, novel TME acid-responsive liposomes for co-delivery of IRI and 398 (IRI&398-s-LPs) were developed, in which the rapid release of both drugs could be triggered under acidic conditions. Notably, a CT-26/3T3 cell co-culture system was used to mimic the real TME both in vitro and in vivo. Cellular immunofluorescence revealed that IRI&398-s-LPs could efficiently decrease the activation of CAFs. In vitro cytotoxicity evaluation demonstrated that IRI&398-s-LPs exhibited higher cytotoxicity than the other liposomal formulations in the CT-26 and CT-26/3T3 cell co-culture system. In vivo NIRF imaging showed that the IRI&398-s-LPs could increase drug accumulation in the tumor sites. Furthermore, IRI&398-s-LPs not only presented superior in vivo anti-tumor activity in CT-26 bearing BALB/c mice, but also enhanced the effect in CT-26/3T3 cell bearing mice with decreased collagen and CAF biomarker expression. Furthermore, IRI&398-s-LPs also presented superior anti-metastatic efficiency in a lung metastasis model. These results indicated that this combinational strategy for eliminating both tumor cells and CAFs provides a new approach for cancer therapy, and the prepared TME-responsive liposomes for co-delivery of drugs hold promising clinical application prospects.

The intracellular chloride channel 4 (CLIC4) activates systemic sclerosis fibroblasts.

Tissue fibrosis in SSc is driven by active fibroblasts (myofibroblasts). Previous studies have shown the intracellular chloride channel 4 (CLIC4) mediates the activation of cancer-associated fibroblasts. In this study we investigated the role of CLIC4 in SSc fibroblast activation. Fibroblasts were obtained from full thickness skin biopsies from SSc patients (early-diffuse). RNA and protein were collected from the fibroblasts and CLIC4 transcript and protein levels were assessed by qPCR and western blot. SSc patient fibroblasts were treated with the chloride channel inhibitors nitro-2-(3-phenylpropylamino)benzoic acid and indyanyloxyacetic acid 94. CLIC4 was expressed at significantly higher levels in SSc patients' fibroblasts compared with healthy controls, at both the transcript (3.7-fold) and protein (1.7-fold) levels. Inhibition of the TGF-β receptor and its downstream transcription factor SMAD3 led to a reduction in CLIC4 expression, confirming this pathway as the main driver of CLIC4 expression. Importantly, treatment of SSc fibroblasts with known pharmacological inhibitors of CLIC4 led to reduced expression of the myofibroblast markers collagen type 1 and α-smooth muscle actin, inferring a direct role for CLIC4 in disease pathogenesis. We have identified a novel role for CLIC4 in SSc myofibroblast activation, which strengthens the similarities of SSc fibroblasts with cancer-associated fibroblasts and highlights this channel as a novel target for therapeutic intervention.

Dysregulation of Transcription Factor Activity During Formation of Cancer-Associated Fibroblasts.

The reciprocal interactions between cancer cells and the quiescent fibroblasts leading to the activation of cancer-associated fibroblasts (CAFs) serve an important role in cancer progression. Here, we investigated the activation of transcription factors (TFs) in prostate fibroblasts (WPMY cell line) co-cultured with normal prostate or tumorous cells (RWPE1 and RWPE2 cell lines, respectively). After indirect co-cultures, we performed mRNA-seq and predicted TF activity using mRNA expression profiles with the Systems EPigenomics Inference of Regulatory Activity (SEPIRA) package and the GTEx and mRNA-seq data of 483 cultured fibroblasts. The initial differential expression analysis between time points and experimental conditions showed that co-culture with normal epithelial cells mainly promotes an inflammatory response in fibroblasts, whereas with the cancerous epithelial, it stimulates transformation by changing the expression of the genes associated with microfilaments. TF activity analysis revealed only one positively regulated TF in the RWPE1 co-culture alone, while we observed dysregulation of 45 TFs (7 decreased activity and 38 increased activity) uniquely in co-culture with RWPE2. Pathway analysis showed that these 45 dysregulated TFs in fibroblasts co-cultured with RWPE2 cells may be associated with the RUNX1 and PTEN pathways. Moreover, we showed that observed dysregulation could be associated with FER1L4 expression. We conclude that phenotypic changes in fibroblast responses to co-culturing with cancer epithelium result from orchestrated dysregulation of signaling pathways that favor their transformation and motility rather than proinflammatory status. This dysregulation can be observed both at the TF and transcriptome levels.

Abstract PO-049: Single-cell transcriptomic analysis reveals interleukin-1 inhibition suppresses inflammatory cancer-associated fibroblast signaling and improves the immune response in pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is resistant to immune checkpoint blockade due to its intrinsic immunosuppressive properties. The fibroinflammatory stroma, particularly inflammatory cancer associated fibroblasts (CAFs), are a key determinant of the immune microenvironment in PDAC, however therapies aimed at broad-based targeting of PDAC stroma have been decidedly unsuccessful. Recently, interleukin-1α (IL-1) has emerged as a critical mediator of CAF activation and orchestrator of immune suppression in PDAC. We sought to explore the effects of IL-1 blockade on reprogramming the immune microenvironment and enhancing susceptibility to checkpoint inhibition through suppression of inflammatory stromal signaling in PDAC. Methods: Ptf1acre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice were treated with the recombinant IL-1 receptor antagonist (anakinra, 50 mg/kg IP BID) or vehicle control. Single-cell cDNA library generation was performed using the 10× Chromium System. Expression matrices were processed using the R package Seurat 3.0 and clustered using AUC-based scoring algorithm. Immunophenotyping experiments were performed on single-cell suspensions using the Cytoflex platform. For survival studies, PKT mice were treated with vehicle, anti-PD1 antibody (200 μg/twice weekly), anakinra, or combination until moribund. Tissues were further processed for downstream qPCR and histologic analyses. Human pancreatic stellate cell (hPSC) and immortalized CAF cell lines procured from patient-derived xenografts were utilized for in vitro mechanistic studies. Results: Single-cell analysis revealed that anakinra treatment in PKT mice reduced levels of numerous pro-inflammatory CAF cytokines, including Cxcl1 and Il6, within CAF subclusters. Confirmatory qPCR of bulk tumor samples revealed a significant reduction in Cxcl1 and Il6 gene transcription and a global reduction in stromal fibrosis with anakinra treatment. IL-1 inhibition selectively reduced alternatively-activated macrophage (CD11b+F480+CD206+) and PMN-MDSC (CD11b+Ly6G+) populations within PDAC tumors. Combination treatment of anakinra with anti-PD1 antibody reduced tumor weight, increased intratumoral levels of effector memory (CD8+CD44+CD62L-) and activated cytotoxic (CD8+CD107+) T cells, and improved overall survival in PKT mice. In vitro studies demonstrated that conditioned media from PDAC tumor cells significantly activated Cxcl1 and Il6 gene transcription in hPSCs and CAFs in an IL-1α-dependent manner. IL-1-mediated transcription of these cytokines was abrogated by selective inhibition of both the p38 MAPK and NFκB cascades, demonstrating a coordinated effort between these signaling pathways in modulating the IL-1-induced stress response in stromal cells. Conclusions: These findings provide important mechanistic data and compelling pre-clinical evidence to explore IL-1 inhibition in combination with immune checkpoint blockade in PDAC patients. Citation Format: Austin R. Dosch, Samara Singh, Xizi Dai, Siddharth Mehra, Anna Bianchi, Iago De Castro Silva, Supriya Srinivasan, Nagaraj Nagathihalli, Jashodeep Datta, Nipun B. Merchant. Single-cell transcriptomic analysis reveals interleukin-1 inhibition suppresses inflammatory cancer-associated fibroblast signaling and improves the immune response in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-049.

Retraction for Meng M et al., CLIC1 facilitates cancer associated fibroblast activation and gastric cancer progression via integrins/NF-κB pathway.

Background: Gastric cancer (GC) is one of the most common and lethal cancers and the prognosis of GC patients remains very poor. Cancer-associated fibroblasts (CAFs) largely facilitate the progression of GC but the underlying molecular mechanisms remain elusive despite numerous studies. Here, we investigated the role of CLIC1 in CAF activation and GC. Methods: qRT-PCR and western blot were performed to determine expression levels of cytokines, transcription factors and related proteins such as CAF markers. MTT assay was used to examine the proliferation of GC cells while transwell assay and tumorsphere formation assay were done to measure the migration, invasion and stemness of GC cells. Conditioned medium model was used to examine the intercellular communication between CAFs and GC cells. Results: Overexpression of CLIC1 in fibroblasts induced CAF activation and enhanced cell proliferation and migration. Also, CLIC1 up-regulated integrins/NF-κB signaling in CAFs. CLIC1-overexpressed fibroblasts promoted proliferation and migration of GC cells and up-regulated cancer stem cell markers and promoted EMT program of GC cells. IL-6 and IL-8 neutralizing antibodies inhibit the pro-tumor effects of CLIC1-overexpressing fibroblasts on GC cells. Further, knockdown CLIC1 in GC cells suppressed activation of CAF. Conclusions: CLIC1 overexpression activates CAF via up-regulating integrins/NF-κB signaling and activated CAF releases IL-6 and IL-8 to promote multiple malignant phenotypes of GC cells. These results implicate an essential role of CLIC1 in CAF activation and GC progression, which suggests that CLIC1 could serve as a potential target for GC therapy.

The vitamin D analogue calcipotriol promotes an anti-tumorigenic phenotype of human pancreatic CAFs but reduces T cell mediated immunity

The pancreatic tumour stroma is composed of phenotypically heterogenous cancer-associated fibroblasts (CAFs) with both pro- and anti-tumorigenic functions. Here, we studied the impact of calcipotriol, a vitamin D3 analogue, on the activation of human pancreatic CAFs and T cells using 2- and 3-dimensional (2D, 3D) cell culture models. We found that calcipotriol decreased CAF proliferation and migration and reduced the release of the pro-tumorigenic factors prostaglandin E2, IL-6, periostin, and leukemia inhibitory factor. However, calcipotriol promoted PD-L1 upregulation, which could influence T cell mediated tumour immune surveillance. Calcipotriol reduced T cell proliferation and production of IFN-γ, granzyme B and IL-17, but increased IL-10 secretion. These effects were even more profound in the presence of CAFs in 2D cultures and in the presence of CAFs and pancreatic tumour cell line (PANC-1) spheroids in 3D cultures. Functional assays on tumour infiltrating lymphocytes also showed a reduction in T cell activation by calcipotriol. This suggests that calcipotriol reduces the tumour supportive activity of CAFs but at the same time reduces T cell effector functions, which could compromise the patients’ tumour immune surveillance. Thus, vitamin D3 analogues appear to have dual functions in the context of pancreatic cancer, which could have important clinical implications.

Cancer-Associated Fibroblasts: Epigenetic Regulation and Therapeutic Intervention in Breast Cancer.

Simple SummaryDrug resistance and insensitivity to treatments are the main challenges in breast cancer therapy. Cancer-associated fibroblasts (CAFs) are heterogeneous stromal cells with prevailing roles in cancer development and progression. Epigenetic alterations are essential in regulating CAF activation and heterogeneity. These modifications are druggable targets that can be reversed using pharmacological interventions. CAFs therefore, have a remarkable potential as a therapeutic target in breast cancer. This review provides an update on the mechanisms of epigenetic modulation in breast cancer and discusses the challenges of translating the optimism of CAF-directed therapies from bench to clinic.Breast cancer is the leading cause of cancer-related mortality in women worldwide. Cancer-associated fibroblasts (CAFs) are a heterogeneous population of cells in the solid tumour microenvironment. These cells are positively linked to breast cancer progression. Breast CAFs can be categorised into distinct subtypes according to their roles in breast carcinogenesis. Epigenetic modifications change gene expression patterns as a consequence of altered chromatin configuration and DNA accessibility to transcriptional machinery, without affecting the primary structure of DNA. Epigenetic dysregulation in breast CAFs may enhance breast cancer cell survival and ultimately lead to therapeutic resistance. A growing body of evidence has described epigenetic modulators that target histones, DNA, and miRNA as a promising approach to treat cancer. This review aims to summarise the current findings on the mechanisms involved in the epigenetic regulation in breast CAFs and discusses the potential therapeutic strategies via targeting these factors.

Lysine demethylase 2A expression in cancer-associated fibroblasts promotes breast tumour growth

BackgroundOur previous study demonstrated that lysine demethylase 2A (KDM2A) enhances stemness in breast cancer cells. This demethylase is also highly expressed in cancer-associated fibroblasts (CAFs). However, its clinical significance is unclear.MethodsThe expression of KDM2A in CAFs was studied using immunohistochemical staining and its association with clinicopathological features and patient’s survival was tested. Overexpression and knockdown strategies were used to investigate KDM2A-regulated genes in fibroblasts. Senescent cells were detected by using β-galactosidase staining. The in vivo tumour-promoting activity of stromal KDM2A was confirmed by animal study.ResultsIncrease of stromal KDM2A is associated with advanced tumour stage and poor clinical outcome in breast cancer patients. Cancer-derived cytokines stimulated KDM2A expression in normal fibroblasts and transformed them into CAFs. Upregulation of KDM2A induced p53-dependent senescence in fibroblasts and enhanced the release of cytokines, which reciprocally promoted cancer cell proliferation. Additionally, KDM2A upregulated programmed death-ligand 1 (PD-L1) expression via transcriptional activation in fibroblasts. Knockdown of KDM2A completely abolished the tumour-promoting activity of CAFs on breast tumour growth in vivo and diminished PD-L1 expression in the stroma of tumour tissues.ConclusionsStromal KDM2A plays an oncogenic role in breast cancer and inhibition of KDM2A reduces fibroblast senescence and suppresses tumour growth.